Water-soluble rhodamine dye conjugates

Abstract

The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

1. A rhodamine dye or a salt thereof, comprising a rhodamine-type parent xanthene ring having attached to the xanthene C9 carbon a phenyl group that is further substituted with an ortho carboxy or ortho sulfonate group or a salt thereof, one to three substituted or unsubstituted aminopyridinium groups and a substituted or unsubstituted alkylthio, arylthio, or heteroarylthio group, said rhodamine dye optionally including one or more linking moieties. 
     
     
       2. The rhodamine dye of  claim 1  which comprises the structure: 
       
         
           
           
               
               
           
         
         wherein:
 n is 1, 2, or 3 
 Y is a rhodamine-type parent xanthene ring attached to the illustrated phenyl group at the xanthene C9 carbon; 
 each R is independently selected from the group consisting of (C 1 -C 6 ) alkyl and heteroalkyl, (C 5 -C 20 ) aryl and heteroaryl, (C 6 -C 26 ) aryalkyl and heteroalklyl, (C 5 -C 20 ) arylaryl and heteroaryl-heteroaryl, or when taken together, R is (C 4 -C 10 ) alkydiyl, (C 4 -C 10 ) alkyleno, heteroalkyldilyl and heteroalkyleno; 
 S is sulfur; 
 Z is (C 1 -C 12 ) alkyl, (C 1 -C 12 ) alkyl substituted with one or more of the same or different W 1  groups (C 5 -C 20 ) aryl and heteroaryl, and (C 5 -C 20 ) aryl and heteroaryl substituted with one or more of the same or different W 2  groups; 
 W 1  is selected from the group consisting of —X, —R, ═O, —OR, —SR, ═S, —NRR, ═NR, —CX 3 , —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO 2 , ═N 2 , —N 3 , —S (O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR; 
 W 2  is selected from the group consisting of —R, —OR, —SR, —NRR, —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O − , —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR; 
 Each x is independently a halogen; and 
 Y or Z is optionally substituted with L where L is a bond or a linker. 
 
       
     
     
       3. A labeled rhodamine dye-polypeptide conjugate comprising the rhodamine dye of  claim 1  and a polypeptide, wherein the polypeptide is selected from the group consisting of a peptide, a protein, and an antibody. 
     
     
       4. A method of detecting a rhodamine dye-antibody conjugate, in which said conjugate is a rhodamine dye-antibody conjugate according to  claim 3 , comprising the steps of:
 (a) binding the conjugates to a peptide or protein, and 
 (b) detecting the rhodamine dye-antibody conjugate bound to the peptide or protein. 
 
     
     
       5. The method of  claim 4  in which the conjugate is bound to the peptide or protein in the presence of a second antibody specific for binding said peptide or protein. 
     
     
       6. The method of  claim 5  in which the second antibody is bound to a solid bead or particle.